New monoclonal antibodies which identify the glycoprotein carrying the ca125 epitope

ABSTRACT

Hybridomally produced monoclonal antibodies specifically immunoreactive with the glycoprotein carrying the CA 125 epitope. Monoclonal antibodies recognize both high and low molecular weight subunits of the antigen, and identify the antigen in the cytoplasm and the extracellular matrix of CA 125 producing cells. An immunoassay for the detection of CA 125 utilizing the monoclonal antibodies is described.

[0001] The invention described herein was supported by NationalInstitute of Health Grant #ROICA40406.

BACKGROUND OF THE INVENTION

[0002] The present invention relates to the identification and use ofmonoclonal antibodies specific for the glycoprotein antigen complexcarrying the CA 125 epitope.

[0003] CA 125 is an antigenic determinant or epitope located on thesurface of ovarian tumor cells with essentially no expression in normaladult ovarian tissue. However, CA 125 is expressed on the cell surfaceof tumor cells in culture and on ovarian tumor lesions. Significantly,CA 125 is elevated in the sera of patients (>90%) with ovarianadenocarcinoma. In fact, CA 125 is regularly detected on the tumor cellsurface and in the serum of patients with serous cystadenocarcinoma ofthe ovary (>95%). Expression of this antigen occurs less frequently inendometrial and clear cell carcinomas, and essentially no expression isdetected in mucinous cystadenocarcinomas. Although not exclusively foundin the blood of these patients, CA 125 has been detected in the sera ofa significant percentage of patients with pancreatic carcinoma(approximately 50%) and with liver and colon carcinoma (approximately22-32%).

[0004] The presence of CA 125 in high concentrations in the serum ofpatients with ovarian adenocarcinoma has been widely used by health careproviders in the treatment and management of such patients. Although CA125 is not specific for ovarian carcinoma, there is, nonetheless, adirect correlation between the presence of CA 125 and disease statusi.e., progression, regression, or no change. Since almost all ovariancancer patients receive extensive chemotherapy, CA 125 is used as anindicator of a disease free state. Further, increased serumconcentrations of CA 125 precede clinical diagnosis of recurrent diseaseby a period of approximately one to four months. As a result, assay ofthis tumor marker is becoming a standard diagnostic tool in monitoringovarian cancer patients.

[0005] As previously described, CA 125 is not an exclusive product ofovarian cancer cells. Like many other tumor markers, CA 125 is alsoexpressed normally in early fetal development. For example, Kabawat etal. (Int J Gynecol Pathol, 2:275-85 (1983)) demonstrates the presence oftraces of CA 125 in fetal tissues; the antigen was localized to theamnion and derivatives of muellerian epithelium, and coelomic epithelium(including the peritoneum, pleura and pericardium). In adult tissues,Kabawat et al. and Hardardottir et al. (Am J Obstet Gynecol,163:1925-1931 (1990)) report that the monoclonal antibody, OC 125,reacts with the epithelium of the fallopian tube, endometrium, andendocervix, and is also expressed in the apocrine sweat glands and themammary glands. Hardardottir et al. further discloses the presence of CA125 antigen during fetal development in notochord, myocardium,pericardium, the mesonephric duct, the vitelline and allantoic ducts, aswell as the amnion and periderm. Elevations of serum CA 125 in patientswith endometriosis, during menses, and in early gestation furtherdemonstrates expression of this antigen during normal growth anddevelopment. The abundance of the antigen in breast milk, benign ovariancyst fluid, and amniotic fluid further implicate CA 125 in normal cellgrowth and development.

[0006] Little is known of the structure of the CA 125 antigen or themetabolic regulation or expression of this antigen in either normal orneoplastic tissues. Current state of the art discloses that CA 125 ispart of a large molecular weight mucin-like glycoprotein complex, whichcan be resolved to a 200 kd-250 kd species on SDS acrylamide oragarose-acrylamide gels. Based on the presence of sugar residues,buoyant density studies, and lectin binding properties, the CA 125antigen is thought to contain a carbohydrate component. However, theantigenic epitope that is recognized by the monoclonal antibody, OC 125,is considered to be peptidic in nature, because of its sensitivity toproteases like trypsin and V8 protease and its relative stability toglycosidases.

[0007] It would be highly desirable to gain a better understanding ofthe structure and function of the CA 125 antigen, especially through thedevelopment of new reagents to map protein domains. The development ofnew reagents such as monoclonal antibodies would likely provide a basisfor new immunoassays with improved sensitivity and specificity for thedetection of CA 125 antigen.

SUMMARY OF THE INVENTION

[0008] The present invention provides hybridomally produced monoclonalantibodies that are specifically immunoreactive with the glycoproteincarrying the CA 125 epitope. The monoclonal antibodies have a bindingaffinity for a high molecular weight (˜>200 kd) subunit and a lowermolecular weight subunit (68 kd) of the CA 125 antigen. These antibodiesrecognize the antigen in the cytoplasm and the extracellular matrix ofCA 125 producing cells. The present invention contemplates the use ofthe monoclonal antibodies in improving the assay and immunohistologicdetection of the CA 125 antigen. Since the antibodies recognize the cellsurface component of tumor cells, it is further anticipated that theantibodies would provide a means of delivering imaging isotopes andchemotherapeutic agents to tumor cells, thereby enhancing the diagnosisand treatment of neoplasms.

[0009] In one embodiment of the present invention, a monoclonal antibodyspecifically immunoreactive with the glycoprotein carrying the CA 125epitope is identified as having a binding affinity for a subunit of theCA 125 epitope having a molecular weight of greater than approximately200k daltons.

[0010] In another embodiment, a monoclonal antibody specificallyimmunoreactive with the glycoprotein carrying the CA 125 epitope isidentified as having a binding affinity for a subunit of the CA 125epitope having a molecular weight of approximately 68k daltons.

[0011] In a further embodiment, a monoclonal antibody specificallyimmunoreactive with the glycoprotein carrying the CA 125 epitope andidentified as M2 is located predominantly in the cytoplasm of CA 125producing cells.

[0012] In yet another embodiment, a monoclonal antibody specificallyimmunoreactive with the glycoprotein carrying the CA 125 epitope andidentified as M11 is located predominantly in the extracellular matrixof CA 125 producing cells.

[0013] Additional features and advantages of the present invention arefurther described, and will be apparent from the presently preferredembodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] A more complete appreciation of the invention and many of theattendant advantages thereof will be readily obtained as the samebecomes better understood by reference to the following detaileddescription when considered in connection with the accompanyingdrawings.

[0015]FIG. 1 illustrates a Western blot of CA 125 rich (22,000 U/ml)ascites fluid. Lanes 1-6 were probed with monoclonal antibodies M2, M11,M6, M1, M3 and control mouse ascites, respectively.

[0016]FIG. 2 illustrates immunoprecipitation of ascites fluid CA 125antigen using newly developed monoclonal antibodies followed by westernblotting and probing of individual lanes with homologous monoclonalantibody. Lanes 1-7 were individually probed with M1, M2, M3, M6, M11,M12 and control mouse ascites, respectively.

[0017]FIG. 3 illustrates immunoprecipitation of ascites fluid CA 125followed by western blotting of precipitates as described in FIG. 2, butindividual lanes were probed with ¹²⁵I-OC 125.

[0018]FIG. 4 illustrates immunoprecipitation of CA 125 from ascitesfluid, amniotic fluid, and normal human serum (Lanes 1-3) withmonoclonal antibody M2 (Panel A), M11 (Panel B), and control ascites(Panel C) followed by western blotting and probing with homologousantibodies.

[0019]FIG. 5 illustrates immunolocalization of CA 125 antigen in amnionepithelium (magnification ×100) using M2 (Panel A), M11 (Panel B), M1(Panel C), OC 125 (Panel D), and control mouse ascites (Panel E).

DETAILED DESCRIPTION OF THE PRESENTLY PREFERRED EMBODIMENTS

[0020] The present invention provides new monoclonal antibodies that arespecific for the glycoprotein antigen complex carrying CA 125, anepitope located on the surface of ovarian carcinoma cells. Morespecifically, the present invention provides hybridomas that are capableof producing monoclonal antibodies which exhibit a greater bindingaffinity for the CA 125 antigen than has been previously heretoforeobserved.

[0021] Use of the hybridomally produced monoclonal antibodies of thepresent invention results in improved assay and immunohistologicdetection of the CA 125 antigen, thereby providing the basis for thedevelopment of new, diagnostic immunoassays for the detection andmonitoring of serum CA 125.

[0022] As will be shown in the following detailed embodiments, theimmunizing antigen, CA 125, was derived from ascites fluid and partiallypurified by the method described by O'Brien et al., Am J Obstet Gynecol,155:50-55 (1986). Balb/c mice were administered dosages of the CA 125antigen intraperitoneally at three different times. Following the thirddosage, the spleens were harvested for fusion with mouse myeloma cells,thereby forming hybridomas. The resulting hybridomas were analyzed forantibody activity and found to secrete antibodies specific against theCA 125 antigen.

[0023] Competition studies were then conducted utilizing the 12 positivehybridomas which recognized CA 125 rich test material. These hybridomaswere incubated with ¹²⁵I-OC 125 antibody in a binding assay for the CA125 antigen. As will be shown in the following example, monoclonalantibody, M2, competed effectively for a site similar to or the same asthe OC 125 antibody.

[0024] Similar studies were carried out by preloading individual OC 125beads with CA 125 antigen obtained from normal human serum, tumor serum,ascites fluid, breast milk, and amniotic fluid containing high levels ofCA 125. CA 125 beads were then exposed individually to monoclonalantibodies, M2 or M11, or to a control in the presence of ¹²⁵I-OC 125.These studies revealed that M2 antibody competed effectively with¹²⁵I-OC 125 antibody regardless of the source of the CA 125 antigen.Additional immunohistochemical studies further demonstrated recognitionof the CA 125 antigen by monoclonal antibodies, M2 and M11, within cellsalso recognized by the monoclonal antibody, OC 125.

[0025] By way of example, and not limitation, the following examples anddrawings serve to further illustrate the present invention in itspreferred embodiments.

EXAMPLE I

[0026] This example illustrates the formation of hybridomas, the sourceof monoclonal antibodies specific to the glycoprotein antigen complexcarrying the CA 125 epitope, and the resulting monoclonal antibodies.

[0027] Preparation of Immunizing Antigen

[0028] CA 125 antigen derived from ascites fluid was partially purified(approximately 2000 units/ug protein) using the method described byO'Brien et al., Am J Obstet Gynecol, 155:50-55 (1986). The antigen wasprecipitated with alum according to the procedure of Hudson and Hay,Practical Immunology, Blackwell Scientific Publications, Oxford, p. 10(1976). The resulting precipitate was washed twice with 15 ul volume of0.01 M phosphate buffered saline consisting of 0.15 M NaCl at pH 7.2(hereinafter “PBS”). The pellet was suspended in 50 ul of PBS andmaintained at 4° C. until use.

[0029] Immunization of Balb/c Mice

[0030] Balb/c mice were immunized by intraperitoneal injection of 20 ugof alum precipitated antigen in 100 ul of PBS containing 10⁹ killedbordetella pertussis organisms (obtained from the Michigan Department ofHealth Lansing, Mich.). After 4 weeks, mice were challenged again byintraperitoneal injection of a 20 ug of soluble antigen in PBS. Twoweeks later, the mice were further challenged with 20 ug of solubleantigen by intraperitoneal injection. Three days later, spleens wereharvested for fusion with mouse myeloma cells.

[0031] Formation of and Cloning Hybridomas

[0032] Fusion of immune mouse splenocytes and 8-azo-guanine resistantP3X63-Ag8.653 mouse myeloma cells was performed according to theprocedures described by de St. Groth and Scheidegger, J. Immunol Meth,35:1-21 (1980). Fifty microliter aliquots of hybridomas were transferredto each well of 5 ninety six well tissue culture plates (Flow Labs),which already contained 200 ul HAT medium (10⁻⁴ M hypoxanthine, 1.6×10⁻⁵M thymidine and 4×10⁻⁷ aminopterin) and 3600 mouse peritonealmacrophages. When hybrids developed, the media were screened utilizing adot blot assay as described in the section below. Positive cultures werecloned utilizing the limiting dilution method in 96 well culture plates.After approximately 2-4 weeks of growth, culture media were againscreened and the positive hybrids grown up and recloned.

[0033] Screening for CA 125 Antibodies

[0034] Culture media were screened using a nitrocellulose dot blotprocedure. To this end, ascites fluid with a CA 125 concentration of22,000 U/ml was diluted 1:20 in PBS. 100 ul were dot blotted tonitrocellulose using a Schleicter and Schuell dot blot apparatus. Acontrol ascites with less than 20 U/ml CA 125 diluted 1:20 was alsoblotted to nitrocellulose. All blots were rinsed twice with PBS andplaced in blotto (5% dry milk powder in PBS) overnight.

[0035] Individual dots were cut from the blot and 100 ul of each hybridculture media were added to tubes containing one control dot (low CA125) and one test dot (high CA 125). Dots were incubated for 3 hours atroom temperature at which time 0.5 ml of blotto was added to each tubeand incubated for an additional 10 minutes. All tubes were thenaspirated and the dots were washed twice with 1 ml of PBS. Tubes werethen incubated with a second antibody, goat-anti-mouse-peroxidase (GAMP,Biorad Corp.), which was diluted 1:250. 250 ul were added to each tube.After the second antibody was incubated for 1 hour at room temperature,the tubes were aspirated. The dots were then washed with PBS and thenexposed to horseradish peroxidase color developer (HRP) as described byBiorad Corporation.

[0036] Productive cultures were transferred to 1 ml culture dishes andgrown up with at least 2 changes of media. Cultures were then screenedusing a dot blot assay similar to that described hereinabove, exceptthat purified CA 125 was used as the positive test dot. Normal humanserum (diluted 1:20) was used as the negative control. Screening wasalso carried out by saturating OC 125 beads (Centocor Malvern, Pa.) withCA 125 antigen by exposing the beads to 1 ml of ascites fluid containing22,000 units CA 125/ml overnight at room temperature. The OC 125 beadswere then washed twice with 5 ml of PBS and further incubated for 3hours at room temperature with rabbit antimouse IgG. This was performedin order to saturate any OC 125 antibody not occupied by CA 125 antigen.

[0037] After washing, beads were exposed to culture media for 3 hours atroom temperature. The beads were washed twice PBS and exposed toperoxidase-coupled antibody (GAMP) and incubated for 1 hour at roomtemperature. Beads were then aspirated and exposed to peroxidasesubstrate for color development. Control culture media was used todetermine background levels of staining.

[0038] Competition of New Antibodies for OC 125 Epitope

[0039] Culture media from positive hybrids along with control culturemedia (from non-productive wells) were incubated with I-OC 125 antibodyin a binding assay for CA 125 antigen. OC 125 beads (Centocor kit) wereexposed to 0.4 ml of ascites fluid (22,000 U/ml CA 125) for overnight atroom temperature. Beads were washed twice with 5 ml PBS and exposed to100 ul of ¹²⁵I-OC 125 antibody in the presence of 100 ul of culturemedia from each of he productive hybrids as well as media from twonon-productive hybrids for a further 18 hours at room temperature. Beadswere washed and counted according to kit instructions. Similarcompetition studies were carried out by preloading individual OC 125beads with CA 125 antigen obtained from normal human serum, tumor serum,ascites fluid, breast milk, and amniotic fluid which contained highlevels of CA 125. CA 125 beads were then exposed individually tohybridomally produced monoclonal antibodies, M2 or M11, or to controlculture media in the presence of ¹²⁵I-OC 125 as described above.

[0040] Groups of 6 Balb/c mice were preconditioned with 0.5 mlperitoneal injections of 0.5 ml of pristane (Aldrich Chemical Co.Milwaukee, Wis.) 14 days prior to injection with 10⁶ hybridoma cells.Ascites fluid was harvested approximately 7-9 days after hybridomainjection. Fluid was centrifuged to remove cells and the supernatentcontaining monoclonal antibody was stored frozen (−70 degrees) untilused.

[0041] Immunoprecipitation

[0042] Aliquots (500 ul) of normal human serum, amniotic fluid and CA125 positive ascites fluid were incubated with 100 ul of 1:100 dilutionof each of the test monoclonal antibodies and 1 control mouse ascitesovernight at 4° C. 200 ul of rabbit antimouse IgG agarose beads (BioradCorp.) diluted 1:2 were added to each tube and incubated for 4 hours atroom temperature with agitation. After centrifugation the beads wereaspirated and washed twice with 1 ml buffer (5% albumin in PBScontaining 0.1% azide) and twice with 1 ml of Bis-Tris buffer (20 mM BisTris propane 7.2). The beads were then exposed to 50 ul of 2×electrophoresis sample buffer for 3 minutes at 100° C. The beads werecooled then pelleted at 2000 RPM for 10 minutes and the supernatantremoved for SDS polyacrylamide gel electrophoresis (PAGE).

[0043] Electrophoresis/Transblotting

[0044] SDS page electrophoresis was carried out on 4-20% gradientpolyacrylamide gels according to Laemmli for 4 hours at 30 m amp. Afterelectrophoresis, the gels were soaked in Tris-glycine buffer (24 mMTris-HCI 192 mM glycine, pH 8.3) and transblotted to an immobilon PVDFfilter (Amicon Corp.) using the Tris glycine buffer with 15% (v/v)methanol overnight at 30 volts.

[0045] Western Blotting

[0046] Immobilon filters were incubated for 30 minutes at roomtemperature with blotto followed by two washes in PBS for 10 minuteseach. Blots are then exposed either to mouse ascites monoclonal antibodyat a dilution of 1:100 in PBS or to OC-125-¹²⁵I antibody (Centocor kit)at a 1:10 dilution at 4° C. overnight. The filters were then washed oncewith blotto for 10 minutes followed by two washes in PBS. The blot wasthen exposed either to x-ray film overnight if I¹²⁵-OC 125 was used orto peroxidase coupled second antibody, (GAMP) at 1:250 dilution (BioradCorp.) if mouse monoclonal ascites were used. A second antibodyincubation was carried out at room temperature for 1 hour. Filters werewashed with blotto and PBS as described above. Blots were developedusing HRP color substrate (Biorad Corp.) as described above.

[0047] Immuonohistochemistry

[0048] For immunohistochemical studies, sections (5 um) offormalin-fixed, paraffin-embedded tissues were deparaffinized andrehydrated through serial alcohol baths to water. The hydrated sectionswere treated with 3% hydrogen peroxide for 10 minutes to blockendogenous peroxidase activity followed by exposure to normal goat serumto reduce non-specific background staining. Primary monoclonal antibody,diluted 1:100 in PBS, was added to sections at room temperature andincubated for 30 minutes. Linking antibody and labelling antibody wereapplied to tissue sections according to kit instructions supplied byDako (Santa Barbara, Calif.) 2nd antibody kits. The substrate used forlocalization of antibody binding was 3-amino-9-ethyl-carbazole (2%).Counter staining was achieved with Mayer's hematoxylin. Negative controlslides were prepared in identical fashion using either normal mouseserum or mouse ascites in place of the primary antibody.

[0049] Results

[0050] The initial screening process of hybrid media utilizing low CA125 and high CA 125 dot blot analysis yielded 24 wells with a positivedot blot test. A dot was scored positive when staining was present onthe test dot (+CA 125) and negative on the control dot (low CA 125).Rescreening of these 24 expanded hybrids utilizing both purified CA 125and normal human serum (low CA 125) by dot blot analysis and by OC 125bead binding yielded 12 positive hybrids of CA 125 antibody producinghybridomas as shown in Table I below. Both dot blots and beads weregraded visually on a scale of 1 to 4 for color development. TABLE IScreening of hybridoma media* Culture Antibody Dot Blot Antigen Bead4A11 Ml 3+ 3+ 3D12 M2 3+ 3+ 1C2 M3 3+ 3+ 1B4 M4 2+ 2+ 2H10 M5 3+ 2+ 3D6M6 2+ 2+ 2E12 M7 2+ 2+ 1H8 M8 2+ 2+ 5F12 M9 1+ 1+ 1C11 M10 1+ 1+ 2G10M11 4+ 2+ 2D12 M12 4+ 2+

[0051] Further analysis of the media from the 12 positive wells bycompetition analysis utilizing 125I-OC 125 binding to the CA 125 antigenin competition with the individual antibodies present in the positivewells indicated that media from one well described in Table II below asM2 effectively competed for a site similar to or the same as the OC 125antibody. Control media or other positive media did not compete with¹²⁵I-OC 125 binding to CA 125. TABLE II Screening of hybridomas forantibodies which compete with OC 125 Monoclonal Antibody % ¹²⁵I-OC BoundM1 94 M2 28 M3 89 M4 98 M5 99 M6 94 M7 100 M8 86 M9 100 M10 92 M11 99M12 100 Control 98

[0052] After cloning, six hybrids continued to recognize CA 125. Thesecloned hybridomas were then utilized to produce ascites in pristanetreated Balb/e mice. When M2 antibody was produced as ascites, iteffectively competed with ¹²⁵I-OC 125 regardless of whether the sourceof CA 125 antigen was serum or ascites obtained from tumor patients orthe CA 125 source was obtained from human serum, normal amniotic fluidor breast milk. These results are shown in Table III below. TABLE IIICompetition for CA 125 epitope % ¹²⁵I-OC 125 Bound Source of AntigenCompetition Ascites Amniotic Breast Tumor Normal Human Medium FluidFluid Milk Serum Serum Control 100 100 100 100 100 M11 Antibody 96 10098 98 95 M12 Antibody 0.9 1.3 2.6 1.8 2.3

[0053] Western blot screening of these monoclonal antibodies indicatethat antibodies, designated M2 and M11, recognize high molecularantigenic determinants in excess of 200k daltons as indicated in Lane 1and Lane 2 of FIG. 1. Electrophoresis patterns of nonpurified CA 125often give a smudged appearance after blotting. This is due to theassociation of the antigen with mucin-like high molecular glycoproteinsas depicted in Lanes 1 and 2 of FIG. 1. Low intensity binding by M6 inLane 3 of FIG. 1 is also detectable on the original blot, but is notreadily visible in FIG. 1.

[0054] Immunoprecipitation of tumor ascites CA 125 antigen was carriedout using monoclonal antibodies derived from the six productivehybridomas and the control mouse ascites. This step was followed byelectrophoresis, blotting, and probing with homologous antibodies. Theseresults indicate that monoclonal antibodies, M2 and M11, shown in Lanes2 and 5, respectively, in FIG. 2 recognize multiple high molecularweight antigenic species in excess of 200 k daltons. On the other hand,monoclonal antibody, M1, recognizes a 68 kd subunit, as depicted in Lane1 of FIG. 2, that is distinct and separate from antibodies M2 and M11.

[0055] Immunoprecipitation, electrophoresis, and blotting of a secondset of these antibodies probed with the heterologous ¹²⁵I-OC 125antibody indicates that OC 125 recognizes at least one of the molecularspecies precipitated by both M2 and M11. This is depicted in Lane 2 andLane 5, respectively, in FIG. 3. No other immunoprecipitate includingthe 68 kd recognized by M1 was readily detected with OC 125.

[0056] Immunoprecipitation of antigen from CA 125 rich sources such asascites (22,000 u/ml) and amniotic fluid (2,000 u/ml) as well as CA 125deficient sources such as normal human serum (<10 units CA 125/ml)further demontrate recognition by M2 and M11 of the CA 125 antigen. Thisis illustrated in FIG. 4, Panels A and B. Immunoprecipitates of ascitesfrom non-immunized mice are shown in Panel C, FIG. 4. Additionally, Lane1 in each Panel is ascites immunoprecipitate (rich in CA 125), andclearly indicates that M2 (Panel A) recognizes high molecular weightantigen from this source. M2 shows only faint bands for CA 125 antigenderived from the less abundant source of CA 125 amniotic fluid (see Lane2, Panel B). Essentially no antigen can be detected inimmunoprecipitates from normal human serum as shown in Lane 3, Panel B.Similarly, probing with M11 confirms that ascites provides a rich sourceof CA 125, while amniotic fluid serves as a much poorer source of theantigen. M11 did not bind immunoprecipitates of normal human serum (seeLanes 1-3, Panel B).

[0057] On the other hand, the control ascites contained no antibodycapable of recognizing CA 125 from all sources of the antigen asillustrated in Panel C of FIG. 4. Bands at 50 kd and approximatley 30 kdrepresent mouse IgG heavy and light chains recognized by the goatanti-mouse second antibody.

[0058] Evaluation of antibodies by immunohistochemical localization inknown CA 125 positive tissues further indicates recognition by theseantibodies of antigen localized within cells also recognized by themonoclonal antibody OC 125. OC 125 has previously been shown todistinctly stain the single epithelial cellular layer of the amnioticmembrane. As illustrated in FIG. 5, comparative staining of monoclonalantibodies M2 (Panel A), M11 (Panel B), M1 (Panel C), and OC 125 (PanelD) along with a mouse ascites control (Panel E) demonstrate the specificlocalization of these antibodies to the amnion. Distribution of antibodywithin the amniotic epithelial cells is more evident with M2 (Panel A)and M1 (Panel C), while extracellular or glycocalyx localization of theantibodies is evident with M11 (Panel B) and OC 125 (Panel D).

[0059] Expression of CA 125 during fetal development on the epitheliumof mullerian duct derivatives (viz the cervix, the corpus and fallopiantube) and on derivatives of coelomic epithelium (peritoneum, pleura andthe pericardium) has been documented by Kabawat et al. Expression of theantigen on the developmental sites is maintained throughout adulthood,resulting in the presence of CA 125 in all non-tumor bearing adults.More recently, the inventor and other investigators described thepresence of abundant levels of CA 125 in amniotic fluid, and it is nowclear that both the amnion and the fetal periderm are major sources ofCA 125 during gestation. It is, therefore, somewhat surprising that CA125 is a useful marker for ovarian cancer in view of the presence of theantigen in abundance in normal tissues. Antigen produced by normaltissues may account for the low but detectable levels of CA 125 whichare measurable in both men and women. The large quantities of CA 125present in the endometrium and cervix are normally secreted to theoutside without access to the circulation. In special cases, for examplein patients with endometriosis, during menses, and during embryoimplantation and early gestation, CA 125 gains access to the circulationthrough disruption of the endothelium and results in elevated CA 125levels. In patients with ovarian carcinoma, access to the circulation inmeasurable amounts occurs relatively late in the disease process.Therefore, most patients with ovarian carcinoma are diagnosed in theadvanced stages (e.g. III and IV) when elevated levels of CA 125 mightbe expected. In fact, at a second look laparotomy when foci of diseaseare less than 2 cm in size, CA 125 is frequently not elevated ordetected in the blood.

[0060] It is apparent from the studies described herein that the newlyidentified monoclonal antibodies exhibit the following: 1) a higheraffinity for the CA 125 glycoprotein, thereby providing the basis for anassay system which could detect relatively low levels of serum CA 125;2) are capable of recognizing the cell surface of tumor cells, therebyproviding a means of radioisotope imaging or delivering therapeuticagents for diagnostic and treatment purposes; and 3) are able torecognize individual domains of the CA 125 molecule for mapping of thebiologically active sites of the CA 125 molecule, thereby providinginsight into the normal functions of the CA 125 antigen.

[0061] One of the newly identified monoclonal antibodies, M2, competeswith OC 125 for binding to the CA 125 antigen. It recognizes largemolecular weight subunits of CA 125 similar to OC 125 and similar to thepurified subunits described by Davis et al., Cancer Res, 46:6143-6148(1986). M11, a second monoclonal antibody, does not compete for the OC125 binding site, but does immunoprecipitate multiple CA 125 subunitssimilar to M2. OC 125 recognizes a major subunit of those precipitatedby M2 and M11. Even though these two antibodies recognize similar CA 125subunits, immunolocalization of the antigen using the amnion as a testsystem indicates a significant difference in cellular distribution ofdeterminants recognized by these two antibodies. In fact, M2 is almostentirely localized within the amniotic epithelium and is relativelyevenly distributed throughout the cytoplasm. In contrast, M11 is almostexclusively localized to the extracellular glycocalyx matrix and appearsto have a much lower recognition of the intracellular antigen than M2.Antibody recognition of antigen localized to the extracellular matrix isof interest because drug delivery or imaging systems are best directedwith antibodies recognizing the cell surface.

[0062] On the other hand, cytoplasmic recognition of the antigen is mostimportant for antibodies which might be used to screen cDNA libraries,where antibodies which recognize protein espitopes with no posttranslational modifications are desired. Both the M1 antibody whichrecognizes a unique 68 kd subunit of the CA 125 molecule and the M2antibody which recognizes high molecular weight subunits of the CA 125molecule are localized in the cytoplasm. The availability of these newantibodies will indeed improve our ability to; (a) further define the CA125 molecule and its constituent domains; (b) screen and potentiallyidentify the CA 125 gene(s); (c) facilitate direct immunopurification ofthe CA 125 antigen; (d) further enhance our ability to detect andspecify tumor CA 125 antigen in patient sera; and (e) direct chemotoxicagents or imaging isotopes to the surface of peritoneal tumor cells.

[0063] It should be understood that various changes and modifications tothe presently preferred embodiments described herein will be apparent tothose skilled in the art. Such changes and modifications can be madewithout departing from the spirit and scope of the present invention,and without diminishing its attendant advantages. It is thereforeintended that such changes and modifications be covered by the appendedclaims.

I claim:
 1. A monoclonal antibody specifically immunoreactive with theglycoprotein carrying the CA 125 epitope for use in the detection ofhuman ovarian carcinoma.
 2. A monoclonal antibody specificallyimmunoreactive with the glycoprotein carrying the CA 125 epitope whereinsaid antibody is identified as M2.
 3. A monoclonal antibody specificallyimmunoreactive with the glycoprotein carrying the CA 125 epitope whereinsaid antibody is identified as M11.
 4. A monoclonal antibodyspecifically immunoreactive with the glycoprotein carrying the CA 125epitope wherein said antibody is identified as M1.
 5. A monoclonalantibody specifically immunoreactive with the glycoprotein carrying theCA 125 epitope wherein the antibody has a binding affinity for a subunitof the CA 125 epitope having a molecular weight of greater thanapproximately 200k daltons.
 6. A monoclonal antibody specificallyimmunoreactive with the glycoprotein carrying the CA 125 epitope whereinthe antibody has a binding affinity for a subunit of the CA 125 epitopehaving a molecular weight of approximately 68k daltons.
 7. Themonoclonal antibody of claim 2, wherein M2 has a binding affinity for asubunit of the CA 125 epitope having a molecular weight of greater thanapproximately 200k daltons.
 8. The monoclonal antibody of claim 2,wherein M2 is located predominantly in the cytoplasm of CA 125 producingcells.
 9. The monoclonal antibody of claim 3, wherein M11 has a bindingaffinity for a subunit of the CA 125 epitope having a molecular weightof greater than approximately 200k daltons.
 10. The monoclonal antibodyof claim 3, wherein M11 is located predominantly in the extracellularmatrix of CA 125 producing cells.
 11. The monoclonal antibody of claim4, wherein M1 has a binding affinity for a subunit of the CA 125 epitopehaving a molecular weight of approximately 68k daltons.
 12. A hybridomacell line capable of producing antibody, M2, specific for theglycoprotein carrying the CA 125 epitope.
 13. A hybridoma cell linecapable of producing antibody, M11, specific for the glycoproteincarrying the CA 125 epitope.
 14. A hybridoma cell line capable ofproducing antibody, M1, specific for the glycoprotein carrying the CA125 epitope.
 15. An immunoassay method utilizing monoclonal antibody,M2, to assay for test material comprising glycoprotein carrying the CA125 epitope, comprising the steps of: contacting a test materialcomprising glycoprotein carrying the CA 125 epitope with the monoclonalantibody M2; and determining the presence of the CA 125 epitope in thetest material.
 16. A immunoassay method utilizing monoclonal antibody,M11, to assay for test material comprising glycoprotein carrying the CA125 epitope, comprising the steps of: contacting a test materialcomprising glycoprotein carrying the CA 125 epitope with the monoclonalantibody M2; and determining the presence of the CA 125 epitope in thetest material.
 17. An immunoassay method utilizing monoclonal antibody,M1, to assay for test material comprising glycoprotein carrying the CA125 epitope, comprising the steps of: contacting a test materialcomprising glycoprotein carrying the CA 125 epitope with the monoclonalantibody M2; and determining the presence of the CA 125 epitope in thetest material.
 18. A monoclonal antibody specifically immunoreactivewith the glycoprotein carrying the CA 125 epitope for use in deliveringtherapeutic agents to tumor cells.
 19. A monoclonal antibodyspecifically immunoreactive with the glycoprotein carrying the CA 125epitope for use in directing imaging agents to tumor cells.
 20. Amonoclonal antibody specifically immunoreactive with the glycoproteincarrying the CA 125 epitope for use in detecting CA 125 in paraffinfixed secretions of human tissues.